- Email: [email protected]
In vivo and in vitro studies of 15α-hydroxyprogesterone in the human placenta
239
IN VIVO AND -Iif\rITRO STUDIES0.c~~c+HYLROXYPROG~TERON~ -I:?WI RUMAN PLtMXJnA 0, L. Tulp and C. I. Meeker MetabolicUnit, Departmentof Medicine,and Departmentof Obstetricsand Gynecology, Universityof VermontCollegeof Medicine, Durlington,Vermont Received: 5/b/76 ARSTRACT Studieswere conductedto determinethe fate of 15c-hydroxyprogesteronein human placentaltissue. Tritiated15c-hydroxyprogesterone was perfusedthroughnormalhuman placentas_In situ at the time of Cesarean sectionand incubatedwith a 10,000x g microeomalsupernateof the placentain vitro. In both systemsthe substrate,but no additional metaboliteswere identified, These findingsindicatethat i5a-hydroxyprogesterone is not metabolizedduring its passagein the human term placenta,and suggeststhat becauseof its fetal originclinical measurementsof 15o-hydroxyprogesterone may providea valuableindex to the statusof fetal viability. INTRODUCTION The steroid15c-hydroxyprogesterone has been isolatedfrom human pregnancyurine by Giannopoulos and Solomon(l),where it appearsin both free and conjugatedform. The quantitiesof steroidisolatedincreasedfrom 3 ug. per day late in the first trimesterto over 30 pg. per day near term. The steroidis uniquelyof fetal origin,and at present Its biologicfunctionis unknown. Tweitand Kagawa (2) have reportedpotentanti-mineralocorticoid activitiesof several15-oxyprogesterones. Rased largelyon the structural similaritybetween15a-hytiroxyprogesterone and severalof these 15-oxyprogesterone compounds,it has been proposedthat the physiologic role of 15a-hydroxyprogesterone may be relatedto a similarmineralocorticoidblockingactivity(1). Severalother hydroxysteroid dehydrog-
enaae systemshave been found in trophoblastic tissue (3-5),
and it
thereforeseemedappropriateto determineif l+hydroxyprogesterone compound could undergofurthertransformation to a 15-oxyprogesterone of greaterantimineralocorticoid activityin placentaltissue, I~i.Zl.'XODS Organicsolventswere of certifiedACS purityand were redistilled prior to use, The commerciallyavailablereferenceand.carriersteroids The [7&!-J-150~hydroxyprogesterone were obtainedfrom Steraloids,Inc. was providedby Dr. S. Solomon,McGill Universityand Royal Victoria HospitalMontreal,Quebec,and had a specificactivityof 2.6 mCi/mmol. All sampleswere countedin a PackardTri Carb Liquid Scintillation Counterwith tritiumefficiencyof 35;;. Sufficientcountingtime was allowed to insurea countingerror of less than 5%. A total of 5.6 and 8.4 uCi of [73H]-lSor-hydroxyprogesterone was perfusedthroughthe placentasof two subjectsin situ (experimentsII and III) at the time of Cesareansection,and 12 xperfused througha glass apparatusmaintainedat 37O C -in vitro (exp. I). Fifty mg. of non-isotopic15c-hydroxyprogesterone was added to the perfusionin experimentIII. The techniqueof placentalperfusionand the detailsof the methodsfor extraction,purificationand identification of metabolites have been describedelsewhere(6). The method is summarizedon the flow diagram in Figure I. The perfusatewas collectedunder absolute PICURE I XZTiiOD FM4 DIAGRAM PlacentalPerfusion Perfusate
Placenta
ikternalUrine
4 day collection Romogenizedwith 5::; frozen ** dextrosein water Total radioactivity Acetoneprecipitation Acetoneprecipitation determined of protein of protein Counter-current distribution Ethyl acetate &hyl acetate extraction extraction* S3, !iZOwash, Dry *** Silicagel gradient TLC - ATLC- B PPC Crystalization Zthanolextraction
+ Nethodsfor placentaand perfusateidenticalfrom here on. ** Not carriedbeyond this point. *** SB = 1;: SodiumBicarbonatewash,
ethanolwhile the placentaltissuewas homogenizedwith 53 dextrosein water, Proteinswere precipitatedwith cold acetone in both instances and the placentalhomogenatethen underwentmultiplecounter-current distributionbetweenaqueous80;:methanoland n-hexane. The final aqueouslayer was reducedto minimalvolume,extractedwith ethylacetate, washedwith 10 nil.aliquotof 1:;sodiumbicarbonateand water, dried over sodiumsulfate,and the dry residuechromatographed on a 100 gram ASTb4'100-200 mesh silicagel column by gradientelutionwith 1-20;; ethanolin nethylenechlorideas outlinedby Engel et al (7). ?l?xflow rate uas adjustedto 80 ml. per hour, and 10 ml. alzu& were collected on a BuchlerFractionCollectoruntil 900-1000ml. of solutehad eluted the column, The specificchromatographic systemsused are outlinedin Table I. Zn the _in vitro studies,10,000x g microsomalhomogenates,were incubatedwith variousconcentrations of 1_5c-hydroxyprogesterone and a TPHH generatingsystemusing the incubationproceduredescribedby Ryan (8,9). P&h homogenatewas equivalentto 16 grams of wet placental tissue, The neutralextractswere appliedto IO gram silica gel columns and developedas outlinedabove by stepwisegradientelution. Ihe radioactive bands elutedfrom these columnswere then processedin a manner identicalto that used for the perfusionexperiments. TABLR I C!3ROXATCGRAPHIC AND CRYSTALIZATION SYSTZXS System SGCG TLC-A
TLC-B PPC CRY-l Q?Y-2 CRY-3
F%ases
i-2@% Ethanolgradient,in Meethylene Chloride n-Hexane : Ethyl Acetate(lrl) Benzene : Zther (1:9) SkellySolveB : Nethanolt H20 (10:9:1) aher-Acetone (1:i) I n-Hexane NethyleneChloride: n-Iiexane Chloroform: SkellySolveB
Developmenttime,hr. 12 2 DoubleDevelopment 2tDoubleDevelopment 100 24 24 24
SGCG = SilicaGel ColumnGradient TLC = Thin layer chromatography~ PPC = Paper partitionchromatography CRY= crystalisation RXSULTS A summaryof the perfusionexperiments, and the initialdistribution of radioactivityis presentedin %.ble II. These data show that in the controlexperimentthe bulk of the radioactivity was recovered, and that approximatelytwo-thirdsrecoveredfrom the _in situ perfusions, Cf the total radioactivity recovered,approximately 40-6% of that
S
242
TIlEOXDS
perfused appeared in the maternal urine during the 4 days following the perfusion, The placental and perfusate material was then extracted several times with ethyl acetate,
?he radioactivity recovered and out-
lined in the lower section of Table II was then applied to silica gel columns, and eluted with increasing concentrations of ct:hanolin methylo? ene chloride. 'Theelution chromatobTarnobtained from the per-F'usate :"A.3Li II ?;?LsYJSIO:I AND SXJ'?RAL;;:x'TRAcT 3ATA A.
Summarv
of Perfusions
Zxperiment Total Su?xtrate I *+ II
III
!bation, ilinutes
3,i5z i*06dpm 7-3-1 I.45 x IO7 dpm '$7 1.88 x IO7 dpn 7- .: plus 5O.w nonisotopic carriers
ml,
3lood Perfused
450 375 400
dli
recovered
all sources
94 69 65
3". Distribution of Radioactivity, dpn Experiment Cord and Placenta Perfusate iiembranes 2.6 x IO6 I 8.4 x IO4 I.3 x IO6 3.0 x IO6 II
111
c.
6.6 x IO5 I.2 x IO6 2.7 x IC6
:~aternal Urine :,Y
Residue
+**
6.0 x IO5 2.9 x IO6 6.1 x 106
2.0 x 106
I.8 x IO5
Xeutral &tract Zecovery Data
Placenta Zxperiment rtesiduewt,gm dpm I
ti ;o
Perfusate wt,(;m dp 2.5~10'g 0.5
i'cSidUo
II
1.9
1.9x106
100
0.5
3,3x106
$3 1'30
IIT
2.3
1.2x1&
100
0.k
3.2~10~
91
* **
15a-hy%droxyprogesterone. Control perfusion, carried out -in vitro in absence of placental material, xi* ;=otalradioactivity remaining in syringe and glassware after perfusion procedure, Total recover& in 4 days post perfusion, ,f the first of the --in situ aerfusions Ls depicted In Figure II. A single peak of rad‘ioactl.vi+,y conLaM.ng 93.6% of the radioactivity elutcd from thj.scolumn in t!?e2,:ethanol _'raction. 'l;eneutral cxt,ra.cts from t5e
controlexperimentand from the perfusateand placentalmaterialfrom the remainingexperiments
was simflarlychromatographed, and in all
columns,an averageof 904%of the radioactivitywas recoveredin a when singlesimilarpeak, Fractionsfrom these peaks of radioactivity, identified,were all found to be i%+vdroxyprogesterone. The specific activityof the steroidrecoveredfrom experimentII was approximately 1% lower than that of the materialperfused,suggestingthe presenceOf Xxtensivechromaa small pool of endogenous15o-hydroxyprogesterone, tographyof the remainingmatorkl recoveredfrom the silica gel columns failed to detectany additionalmetabolites. Orystalization data from one of the perfusionexperimentsis summsrieedin Table III, Melting points for the crystalsfrom the perfusatewere 224-225.7°Cand for the crystalsfrom the placenta222.5-22tl°C, Meltingpointsfor authentic i5cc-hydroxyprogesterone were 225.5-~6,5~~. FIGURE II TH” C?iHOMAToGRAMOF T?iIE Nl3vm.AL EX!fwACT OF !miE
PBWJSATE P%OM EXPE!?IMZNT II ON A100
GM SILICAGEL OOIJJMN
31
Pour _in vitro incubationswere carriedout with 1.3 x IO6 dpm of 3H-~50-h~ox~ogest~~one added to each, Two of the incubationshad 200 VE of non-isotopic
lSe-hjrdroxyprogesterone added as carrier, The
TABLE III CRYSTALIZATIOM DATA F'ORISe-HYDROXYPRCCSSTERO~~FROM ZXPSRIhEi?TIII Specific Activity, dpm/mg Perfusate Placenta. Crystal N.L. Crystal 1830 1420 170 1680 600 1720 1510 1540 610 1440 550
Crystal No. 1 L 3b
i4.L. 110 780 570
third contained 1 mg. of carrier while the fourth served as a control, and contained no placental material, The recovery of the radioactivity in the neutral extracts from these incubations contained an average of 9% of that incubated, Vhen chromatographed on 10 gram silica gel columns as before, single peaks of radioactivity averaging 93% of the material c,hromatographed were isolated in the 3’: ethanol fraction, The peaks of radioactivity were each identified as lja-hydroxyprogesterone. 30 other metabolites were found, and one 2OOug.
Crystalisation data for the control
incubation is summarized in Iable ET. TABLi3I?
CRYSTALIZATIOH DATA, -IN 7ITRO IiJCUBATIOiL’S
Cryst'n 1 L 3b KP
X0.
crystal 4210 3460 2900
Specific Activity, dpm mg Control Substrate, 2OOu crystal ~+I10 Fiti ;z 35io 2270 3400 225-225.7OC
3030 222-223.2Oc
g PI. L . 4020 2300 3490
D1SCLJSS10N Cur failure to identify metabolites in either the -in vivo or & vitro systems studied following an exhaustive chroinatographic search suggests that no significant metabolism occurs during the trans-placental passage of 15cL-hycb-oxyprogesterone, in contrast to earlier speculations,
The biologicfunctionof this steroidremainsunclear,but maY be lated
re-
to the metabolismof other steroids,or may itselfbe a metabolite
et al (11)reof progesterone,Ruse and Solomon(10)and Giannopoulos -ported
that, both the 15 and 16 hydroxylation of progesteroneinfluenced
reductionin the A and 13rings of the steroidnucleus. In addition, they reportedthat the 15~hyclroxygroup influencedthe reductionof the 20-ketoneof progesteronetoward the formationof the 208-alcohol.This observationthat 15~hydroxyprogesterone and other ring-D steroidsmay influencemetabolicbehavioris of interest, The regulatoryfactors which controlsteroidmetabolismare presentlypoorlyunderstood,both in the adult and fetal organism,and the observationthat ring-3 hydroxysteroids may influencesteroidreductionin the adult sug;;ests that a similaraction may be involvedin the fetus, This steroidmay thereforebe implicatedin a regulatoryfunction,eitherin steroid metabolism,or in some other aspect of fetal homeostasis. All peaks of radioactivityelutedfrom the silicagel columnswere examinedfor the presenceof metabolites,but none were isolated. In all experiments,the substrateelutedpredominantly in the 2 to 3% ethanolfraction,consistentwith findingsfrom other laboratories(10). Although15a-hydroxyprogesterone was not metabolizedin the placental tissuesstudied,this does not precludeits' transformation elsewherein promotesome protectiveor the fetus. Should 1%~hydroxyprogesterone re,rulatory effecton fetal tissues,perhapsthese same tissuesare the site of its metabolism, In addition,lack of placentalmetabolismof a steroidwhich is uniquelyelaboratedby the fetus greatlyincreasesits potentialvalue as a measurableindex of fetal well being, Therefore,the measurement
S
246
o,C
TElROIDm
t!-lis s:;eroidin either maternal urine or amniotic fluid may have
[email protected]';clinical application in deternininc viability of the :"etus near
Supported by the U.S. i>ubliciIealthService Grant iio.E*C2732. The authors wish to thank Dr. S. Solomon of KcGill University for providing the 1:~hydroxyprogesteronc used in these studies.
Giannopoulos, G. and Solomon, S., Biochemistry 681226 (1967). Tweit, R. C. and Eqawa, C, L, J, Med. Chem, 3524 (1964). i$,rt,D. IbbK,, J, Endocr, 3_5:2_55 (1966). ;: Billie, A, II.,Cameron, & H, D., Griffiths, I:.and ?&rt, D, Xc?:. 4. J, Zndocr. a:227 (1965). Osinski, 1. A., Xature 187:~~~ (1960). iiieeker, C, I., Stern, M. D,, and Solomon, S., Gzn. J. Biochom. 2: 9:32 (1971). &gel, L. L., Cameron, C. i3.,Stoffyn, A., Alexander, J. A., 7. KLein, O., and 3ofimou, 1:.D., Anal Xochem. 2:114 (1961). Ryan, K. J., Endocrinology Q:392 (195%). %, Zyan, K. J., Biochem Diophfs Acta a:658 (1958). 9. 31s0, J. L., and Solomon, S., Ziochemistry I:1065 (1956). ::: ~iannopoulos, G., Xhavnani, 3. 9., Younglai, Z:.V., and Solomon, S, The Foeto Placental Unit, 3litors A. Pecile and C. Finzi, Lxerpta Ked. Found., Amsterdam, International Symposium, Eilan, Italy, (1960). I.
IN VIVO AND -Iif\rITRO STUDIES0.c~~c+HYLROXYPROG~TERON~ -I:?WI RUMAN PLtMXJnA 0, L. Tulp and C. I. Meeker MetabolicUnit, Departmentof Medicine,and Departmentof Obstetricsand Gynecology, Universityof VermontCollegeof Medicine, Durlington,Vermont Received: 5/b/76 ARSTRACT Studieswere conductedto determinethe fate of 15c-hydroxyprogesteronein human placentaltissue. Tritiated15c-hydroxyprogesterone was perfusedthroughnormalhuman placentas_In situ at the time of Cesarean sectionand incubatedwith a 10,000x g microeomalsupernateof the placentain vitro. In both systemsthe substrate,but no additional metaboliteswere identified, These findingsindicatethat i5a-hydroxyprogesterone is not metabolizedduring its passagein the human term placenta,and suggeststhat becauseof its fetal originclinical measurementsof 15o-hydroxyprogesterone may providea valuableindex to the statusof fetal viability. INTRODUCTION The steroid15c-hydroxyprogesterone has been isolatedfrom human pregnancyurine by Giannopoulos and Solomon(l),where it appearsin both free and conjugatedform. The quantitiesof steroidisolatedincreasedfrom 3 ug. per day late in the first trimesterto over 30 pg. per day near term. The steroidis uniquelyof fetal origin,and at present Its biologicfunctionis unknown. Tweitand Kagawa (2) have reportedpotentanti-mineralocorticoid activitiesof several15-oxyprogesterones. Rased largelyon the structural similaritybetween15a-hytiroxyprogesterone and severalof these 15-oxyprogesterone compounds,it has been proposedthat the physiologic role of 15a-hydroxyprogesterone may be relatedto a similarmineralocorticoidblockingactivity(1). Severalother hydroxysteroid dehydrog-
enaae systemshave been found in trophoblastic tissue (3-5),
and it
thereforeseemedappropriateto determineif l+hydroxyprogesterone compound could undergofurthertransformation to a 15-oxyprogesterone of greaterantimineralocorticoid activityin placentaltissue, I~i.Zl.'XODS Organicsolventswere of certifiedACS purityand were redistilled prior to use, The commerciallyavailablereferenceand.carriersteroids The [7&!-J-150~hydroxyprogesterone were obtainedfrom Steraloids,Inc. was providedby Dr. S. Solomon,McGill Universityand Royal Victoria HospitalMontreal,Quebec,and had a specificactivityof 2.6 mCi/mmol. All sampleswere countedin a PackardTri Carb Liquid Scintillation Counterwith tritiumefficiencyof 35;;. Sufficientcountingtime was allowed to insurea countingerror of less than 5%. A total of 5.6 and 8.4 uCi of [73H]-lSor-hydroxyprogesterone was perfusedthroughthe placentasof two subjectsin situ (experimentsII and III) at the time of Cesareansection,and 12 xperfused througha glass apparatusmaintainedat 37O C -in vitro (exp. I). Fifty mg. of non-isotopic15c-hydroxyprogesterone was added to the perfusionin experimentIII. The techniqueof placentalperfusionand the detailsof the methodsfor extraction,purificationand identification of metabolites have been describedelsewhere(6). The method is summarizedon the flow diagram in Figure I. The perfusatewas collectedunder absolute PICURE I XZTiiOD FM4 DIAGRAM PlacentalPerfusion Perfusate
Placenta
ikternalUrine
4 day collection Romogenizedwith 5::; frozen ** dextrosein water Total radioactivity Acetoneprecipitation Acetoneprecipitation determined of protein of protein Counter-current distribution Ethyl acetate &hyl acetate extraction extraction* S3, !iZOwash, Dry *** Silicagel gradient TLC - ATLC- B PPC Crystalization Zthanolextraction
+ Nethodsfor placentaand perfusateidenticalfrom here on. ** Not carriedbeyond this point. *** SB = 1;: SodiumBicarbonatewash,
ethanolwhile the placentaltissuewas homogenizedwith 53 dextrosein water, Proteinswere precipitatedwith cold acetone in both instances and the placentalhomogenatethen underwentmultiplecounter-current distributionbetweenaqueous80;:methanoland n-hexane. The final aqueouslayer was reducedto minimalvolume,extractedwith ethylacetate, washedwith 10 nil.aliquotof 1:;sodiumbicarbonateand water, dried over sodiumsulfate,and the dry residuechromatographed on a 100 gram ASTb4'100-200 mesh silicagel column by gradientelutionwith 1-20;; ethanolin nethylenechlorideas outlinedby Engel et al (7). ?l?xflow rate uas adjustedto 80 ml. per hour, and 10 ml. alzu& were collected on a BuchlerFractionCollectoruntil 900-1000ml. of solutehad eluted the column, The specificchromatographic systemsused are outlinedin Table I. Zn the _in vitro studies,10,000x g microsomalhomogenates,were incubatedwith variousconcentrations of 1_5c-hydroxyprogesterone and a TPHH generatingsystemusing the incubationproceduredescribedby Ryan (8,9). P&h homogenatewas equivalentto 16 grams of wet placental tissue, The neutralextractswere appliedto IO gram silica gel columns and developedas outlinedabove by stepwisegradientelution. Ihe radioactive bands elutedfrom these columnswere then processedin a manner identicalto that used for the perfusionexperiments. TABLR I C!3ROXATCGRAPHIC AND CRYSTALIZATION SYSTZXS System SGCG TLC-A
TLC-B PPC CRY-l Q?Y-2 CRY-3
F%ases
i-2@% Ethanolgradient,in Meethylene Chloride n-Hexane : Ethyl Acetate(lrl) Benzene : Zther (1:9) SkellySolveB : Nethanolt H20 (10:9:1) aher-Acetone (1:i) I n-Hexane NethyleneChloride: n-Iiexane Chloroform: SkellySolveB
Developmenttime,hr. 12 2 DoubleDevelopment 2tDoubleDevelopment 100 24 24 24
SGCG = SilicaGel ColumnGradient TLC = Thin layer chromatography~ PPC = Paper partitionchromatography CRY= crystalisation RXSULTS A summaryof the perfusionexperiments, and the initialdistribution of radioactivityis presentedin %.ble II. These data show that in the controlexperimentthe bulk of the radioactivity was recovered, and that approximatelytwo-thirdsrecoveredfrom the _in situ perfusions, Cf the total radioactivity recovered,approximately 40-6% of that
S
242
TIlEOXDS
perfused appeared in the maternal urine during the 4 days following the perfusion, The placental and perfusate material was then extracted several times with ethyl acetate,
?he radioactivity recovered and out-
lined in the lower section of Table II was then applied to silica gel columns, and eluted with increasing concentrations of ct:hanolin methylo? ene chloride. 'Theelution chromatobTarnobtained from the per-F'usate :"A.3Li II ?;?LsYJSIO:I AND SXJ'?RAL;;:x'TRAcT 3ATA A.
Summarv
of Perfusions
Zxperiment Total Su?xtrate I *+ II
III
!bation, ilinutes
3,i5z i*06dpm 7-3-1 I.45 x IO7 dpm '$7 1.88 x IO7 dpn 7- .: plus 5O.w nonisotopic carriers
ml,
3lood Perfused
450 375 400
dli
recovered
all sources
94 69 65
3". Distribution of Radioactivity, dpn Experiment Cord and Placenta Perfusate iiembranes 2.6 x IO6 I 8.4 x IO4 I.3 x IO6 3.0 x IO6 II
111
c.
6.6 x IO5 I.2 x IO6 2.7 x IC6
:~aternal Urine :,Y
Residue
+**
6.0 x IO5 2.9 x IO6 6.1 x 106
2.0 x 106
I.8 x IO5
Xeutral &tract Zecovery Data
Placenta Zxperiment rtesiduewt,gm dpm I
ti ;o
Perfusate wt,(;m dp 2.5~10'g 0.5
i'cSidUo
II
1.9
1.9x106
100
0.5
3,3x106
$3 1'30
IIT
2.3
1.2x1&
100
0.k
3.2~10~
91
* **
15a-hy%droxyprogesterone. Control perfusion, carried out -in vitro in absence of placental material, xi* ;=otalradioactivity remaining in syringe and glassware after perfusion procedure, Total recover& in 4 days post perfusion, ,f the first of the --in situ aerfusions Ls depicted In Figure II. A single peak of rad‘ioactl.vi+,y conLaM.ng 93.6% of the radioactivity elutcd from thj.scolumn in t!?e2,:ethanol _'raction. 'l;eneutral cxt,ra.cts from t5e
controlexperimentand from the perfusateand placentalmaterialfrom the remainingexperiments
was simflarlychromatographed, and in all
columns,an averageof 904%of the radioactivitywas recoveredin a when singlesimilarpeak, Fractionsfrom these peaks of radioactivity, identified,were all found to be i%+vdroxyprogesterone. The specific activityof the steroidrecoveredfrom experimentII was approximately 1% lower than that of the materialperfused,suggestingthe presenceOf Xxtensivechromaa small pool of endogenous15o-hydroxyprogesterone, tographyof the remainingmatorkl recoveredfrom the silica gel columns failed to detectany additionalmetabolites. Orystalization data from one of the perfusionexperimentsis summsrieedin Table III, Melting points for the crystalsfrom the perfusatewere 224-225.7°Cand for the crystalsfrom the placenta222.5-22tl°C, Meltingpointsfor authentic i5cc-hydroxyprogesterone were 225.5-~6,5~~. FIGURE II TH” C?iHOMAToGRAMOF T?iIE Nl3vm.AL EX!fwACT OF !miE
PBWJSATE P%OM EXPE!?IMZNT II ON A100
GM SILICAGEL OOIJJMN
31
Pour _in vitro incubationswere carriedout with 1.3 x IO6 dpm of 3H-~50-h~ox~ogest~~one added to each, Two of the incubationshad 200 VE of non-isotopic
lSe-hjrdroxyprogesterone added as carrier, The
TABLE III CRYSTALIZATIOM DATA F'ORISe-HYDROXYPRCCSSTERO~~FROM ZXPSRIhEi?TIII Specific Activity, dpm/mg Perfusate Placenta. Crystal N.L. Crystal 1830 1420 170 1680 600 1720 1510 1540 610 1440 550
Crystal No. 1 L 3b
i4.L. 110 780 570
third contained 1 mg. of carrier while the fourth served as a control, and contained no placental material, The recovery of the radioactivity in the neutral extracts from these incubations contained an average of 9% of that incubated, Vhen chromatographed on 10 gram silica gel columns as before, single peaks of radioactivity averaging 93% of the material c,hromatographed were isolated in the 3’: ethanol fraction, The peaks of radioactivity were each identified as lja-hydroxyprogesterone. 30 other metabolites were found, and one 2OOug.
Crystalisation data for the control
incubation is summarized in Iable ET. TABLi3I?
CRYSTALIZATIOH DATA, -IN 7ITRO IiJCUBATIOiL’S
Cryst'n 1 L 3b KP
X0.
crystal 4210 3460 2900
Specific Activity, dpm mg Control Substrate, 2OOu crystal ~+I10 Fiti ;z 35io 2270 3400 225-225.7OC
3030 222-223.2Oc
g PI. L . 4020 2300 3490
D1SCLJSS10N Cur failure to identify metabolites in either the -in vivo or & vitro systems studied following an exhaustive chroinatographic search suggests that no significant metabolism occurs during the trans-placental passage of 15cL-hycb-oxyprogesterone, in contrast to earlier speculations,
The biologicfunctionof this steroidremainsunclear,but maY be lated
re-
to the metabolismof other steroids,or may itselfbe a metabolite
et al (11)reof progesterone,Ruse and Solomon(10)and Giannopoulos -ported
that, both the 15 and 16 hydroxylation of progesteroneinfluenced
reductionin the A and 13rings of the steroidnucleus. In addition, they reportedthat the 15~hyclroxygroup influencedthe reductionof the 20-ketoneof progesteronetoward the formationof the 208-alcohol.This observationthat 15~hydroxyprogesterone and other ring-D steroidsmay influencemetabolicbehavioris of interest, The regulatoryfactors which controlsteroidmetabolismare presentlypoorlyunderstood,both in the adult and fetal organism,and the observationthat ring-3 hydroxysteroids may influencesteroidreductionin the adult sug;;ests that a similaraction may be involvedin the fetus, This steroidmay thereforebe implicatedin a regulatoryfunction,eitherin steroid metabolism,or in some other aspect of fetal homeostasis. All peaks of radioactivityelutedfrom the silicagel columnswere examinedfor the presenceof metabolites,but none were isolated. In all experiments,the substrateelutedpredominantly in the 2 to 3% ethanolfraction,consistentwith findingsfrom other laboratories(10). Although15a-hydroxyprogesterone was not metabolizedin the placental tissuesstudied,this does not precludeits' transformation elsewherein promotesome protectiveor the fetus. Should 1%~hydroxyprogesterone re,rulatory effecton fetal tissues,perhapsthese same tissuesare the site of its metabolism, In addition,lack of placentalmetabolismof a steroidwhich is uniquelyelaboratedby the fetus greatlyincreasesits potentialvalue as a measurableindex of fetal well being, Therefore,the measurement
S
246
o,C
TElROIDm
t!-lis s:;eroidin either maternal urine or amniotic fluid may have
[email protected]';clinical application in deternininc viability of the :"etus near
Supported by the U.S. i>ubliciIealthService Grant iio.E*C2732. The authors wish to thank Dr. S. Solomon of KcGill University for providing the 1:~hydroxyprogesteronc used in these studies.
Giannopoulos, G. and Solomon, S., Biochemistry 681226 (1967). Tweit, R. C. and Eqawa, C, L, J, Med. Chem, 3524 (1964). i$,rt,D. IbbK,, J, Endocr, 3_5:2_55 (1966). ;: Billie, A, II.,Cameron, & H, D., Griffiths, I:.and ?&rt, D, Xc?:. 4. J, Zndocr. a:227 (1965). Osinski, 1. A., Xature 187:~~~ (1960). iiieeker, C, I., Stern, M. D,, and Solomon, S., Gzn. J. Biochom. 2: 9:32 (1971). &gel, L. L., Cameron, C. i3.,Stoffyn, A., Alexander, J. A., 7. KLein, O., and 3ofimou, 1:.D., Anal Xochem. 2:114 (1961). Ryan, K. J., Endocrinology Q:392 (195%). %, Zyan, K. J., Biochem Diophfs Acta a:658 (1958). 9. 31s0, J. L., and Solomon, S., Ziochemistry I:1065 (1956). ::: ~iannopoulos, G., Xhavnani, 3. 9., Younglai, Z:.V., and Solomon, S, The Foeto Placental Unit, 3litors A. Pecile and C. Finzi, Lxerpta Ked. Found., Amsterdam, International Symposium, Eilan, Italy, (1960). I.